Reducing tetracycline resistance in living cells

ABSTRACT

The present invention provides an improved methodology by which therapeutically to overcome resistance to tetracycline in living cells including bacteria, parasites, fungi, and rickettsiae. The methodology employs a blocking agent such as C5 ester derivatives, or 6-deoxy 13-(substituted mercapto) derivatives of tetracycline, in combination with other tetracycline-type antibiotics as a synergistic combination of compositions to be administered simultaneously, sequentially or concurrently. In another embodiment, certain novel compositions are provided which may be administered alone against, for example, a sensitive or resistant strain of gram positive bacteria such as S. aureus and E. faecalis. The concomitantly administered compositions effectively overcome the tetracycline resistant mechanisms present such that the cell is effectively converted from a tetracycline-resistant state to a tetracycline-sensitive state.

CROSS-REFERENCE

This is a continuation of application Ser. No. 08/232,247 filed on Jun.14, 1994, now U.S. Pat. No. 5,589,470; which is a Continuation-In-Partapplication of application Ser. No. 07/788,693 filed Nov. 6, 1991 nowabandoned, which is a Continuation-In-Part application of applicationSer. No. 07/484,904 filed Feb. 26, 1990, now U.S. Pat. No. 5,064,821issued Nov. 12, 1991, which is a Continuation-In-Part of applicationSer. No. 07/672,323 filed Mar. 20, 1991, now U.S. Pat. No. 5,258,372which is a Continuation-In-Part of application Ser. No. 06/850,843 filedApr. 11, 1986, now U.S. Pat. No. 5,021,407 issued Jun. 4, 1991, which isa Continuation of application Ser. No. 06/442,688 filed Nov. 18, 1982,now U.S. Pat. No. 4,806,529 issued Feb. 21, 1989.

RESEARCH REPORT

The research for the present invention was supported by funds obtainedthrough Tufts University.

FIELD OF THE INVENTION

The present invention concerns therapeutic tetracycline treatment ofliving cells, and is particularly directed to methods and materials foraltering and overcoming resistance to tetracycline within microorganismssuch as bacteria, fungi, rickettsia, and the like.

BACKGROUND OF THE INVENTION

The development of the tetracycline antibiotics was the direct result ofa systematic screening of soil specimens collected from many parts ofthe world for evidence of microorganisms capable of producingbacteriocidal and/or bacteriostatic compositions. The first of thesenovel compounds was introduced in 1948 under the name chlortetracycline.Two years later oxytetracycline became available. The detailedelucidation of the chemical structure of these agents confirmed theirsimilarity and furnished the analytical basis for the production of athird member of this group in 1952, tetracycline. By 1957, a new familyof tetracycline compositions characterized chemically by the absence ofthe ring-attached CH₃ group present in the earlier compositions wasprepared and became publicly available in 1959 under the official namedemeclocycline. Subsequently, methacycline, a derivative ofoxytetracycline, was introduced in 1966; doxycycline became available by1967; and minocycline was in use by 1972. For clarity, for general easeof understanding, and for comparison purposes, these individualtetracycline type agents are structurally compared within Table I below.

                  TABLE I                                                         ______________________________________                                        TETRACYCLINE                                                                   ##STR1##                                                                                                   At Carbon                                       Congener     Substituent(s)   Position Nos.                                   ______________________________________                                        Chlortetracycline                                                                          Cl               (7)                                             Oxytetracycline                                                                            OH, H            (5)                                             Demeclocycline                                                                             OH, H; Cl        (6;7)                                           Methacycline OH, H; CH.sub.2  (5;6)                                           Doxycycline  OH, H; CH.sub.3, H                                                                             (5;6)                                           Minocycline  H, H; N(CH.sub.3).sub.2                                                                        (6;7)                                           ______________________________________                                    

Subsequent to these initial developments, much research effort wasfocused on developing new tetracycline antibiotic compositions effectiveunder varying therapeutic conditions and routes of administration; andfor developing new tetracycline analogues which might prove to be equalor more effective than the originally introduced tetracycline familiesbeginning in 1948. Representative of such developments are U.S. Pat.Nos. 3,957,980; 3,674,859; 2,980,584; 2,990,331; 3,062,717; 3,557,280;4,018,889; 4,024,272; 4,126,680; 3,454,697; and 3,165,531. It will beunderstood that these issued patents are merely representative of therange of diversity of investigations seeking tetracycline andtetracycline analogue compositions which are pharmacologically active.

Historically, soon after their initial development and introduction, thetetracyclines regardless of specific formulation or chemical structurewere found to be highly effective pharmacologically against rickettsiae;a number of gram-positive and gram-negative bacteria; and the agentsresponsible for lymphogranuloma venereum, inclusion conjunctivitis, andpsittacosis. Hence, tetracyclines became known as "broad spectrum"antibiotics. With the subsequent establishment of their in-vitroantimicrobial activity, effectiveness in experimental infections, andpharmacological properties, the tetracyclines as a class rapidly becamewidely used for therapeutic purposes. However, this widespread use oftetracyclines for both major and minor illnesses and diseases leddirectly to the emergence of resistance to these antibiotics even amonghighly susceptible bacterial species both commensal and pathogenic--asfor example pneumococci and Salmonella. The rise oftetracycline-resistant organisms has led not only to a general declinein use of tetracyclines and tetracycline analogue compositions asantibiotics of choice, but has also launched major efforts andinvestigations to uncover the mechanism for tetracycline resistance--inthe hope that some effective means might be developed to overcome theproblem of tetracycline-resistance and thus reestablish thepharmacological value and efficacy of tetracyclines as a whole.

The following represents a current summary of the investigations andknowledge regarding the mechanism of action for tetracyclines inbacteria. The principal site of action for tetracyclines is thebacterial ribosome; at least two different processes appear to berequired for tetracyclines to gain access to the cytoplasm and theribosomes of bacteria. The first process is a passive diffusion of thetetracycline through hydrophilic pores located in the outer cellmembrane. One of these structures is the major outer membrane protein,Omp F in E. coli. The second process involves an energy-dependent activetransport system that pumps all tetracyclines through the innercytoplasmic membrane into the cytoplasm of the cell. In thetetracycline-sensitive cell or organism, once the tetracycline gainsaccess to the interior of the cell, it is able to bind to the ribosomesand inhibit protein synthesis. However, in many tetracycline resistantcells and organisms, an efflux pump system is present which appears tobind the tetracycline molecule and actively transports the tetracyclinemolecule out of the organism into the surrounding environment. Thisactive efflux employs an inner membrane protein designated TET (or Tet)protein which is synthesized in the cell from a gene which is generallyacquired by the organism. Often the gene is present on anextra-chromosomal, autonomously replicating plasmid or a transposon.

Tetracycline resistance is often regulated--that is, inducible bytetracycline. Investigations of active tetracycline efflux systems andthe details of the active efflux mechanism of action have been welldocumented and include the following publications, each of which isexpressly incorporated by reference herein: Chopra et al., J.Antimicrobiol. Chemotherapy 8:5-21 (1981); Levy and McMurry, Biochem.Biophys. Res. Comm. 56:1060-1068 (1974); Levy and McMurry, Nature275:90-92 (1978); McMurry and Levy, Antimicrobial Agents AndChemotherapy 114:201-209 (1978); McMurry et al., Proc. Nat. Acad. Sci.U.S.A. 77:3974-3977 (1980); Ball et al., Biochem. Biophys. Res. Comm.93:74-81 (1980); Curiale and Levy, J. Bact. 151:209-2115 (1982); Mendezet al., Plasmid 3:99-108 (1980); Curiale et al., J. Bact. 157:211-217(1984); and Levy, S. B., Journal of Antimicrobial Chemotherapy 24:1-3(1989).

In addition, a second mechanism of tetracycline resistance for cells isknown and in effect. This resistance mechanism involves a cytoplasmicprotein which protects the intracellular ribosomes from the inhibitoryaction of tetracyclines. This form of tetracycline resistance isdescribed within Burdett, V., J. Bact. 165:564-569 (1986); and Levy, S.B., J. Antimicrob. Chem. 24:1-3 (1989).

With the increased understanding and knowledge regarding the origin andthe mechanisms of tetracycline resistance in various cells andmicroorganisms, active investigations and developments seeking means forovercoming these mechanisms, notably the active efflux system have beenattempted. One successful approach is described within U.S. Pat. No.4,806,529 issued Feb. 21, 1989 --an innovation which is a precursor ofmore recent developments, namely U.S. Pat. No. 5,064,821 issued Nov. 12,1991. Clearly, additional methods and materials for overcomingtetracycline-resistance in bacteria and other organisms are mostdesirable and needed. Substantive advances which additionally overcomethe active efflux system for tetracycline and/or the ribosomalprotection mechanism in the resistant cell would be presently recognizedby the ordinary practitioner in the art as a major asset and innovation.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions fortherapeutically treating a tetracycline-resistant cell and also providesa method for altering a cell from a tetracycline-resistant state into atetracycline-sensitive state. In one preferred embodiment, this methodcomprises the steps of: administering to the cell a predeterminedquantity of at least a first composition selected from the chemicalgroup consisting of a blocking agent which is capable of interactingwith, e.g. binding to, a product of at least one tetracycline resistancedeterminant capable of protecting ribosomes in the cell fromtetracycline's inhibitory activity; and

concomitantly administering to the cell a pre-determined quantity of atleast a second composition selected from the chemical group consistingof tetracycline, tetracycline analogues, and tetracycline derivativeswhich are not said blocking agent. The cell is allowed to preferentiallyreact with the blocking agent.

The unique methodology is able to alter and to converttetracycline-resistant cells or microorganisms intotetracycline-sensitive ones; and, accordingly, to provide a therapeutictreatment for those living subjects, human, animal, and plants, whichhave been previously refractory to a tetracycline therapeutic regimen.

In another embodiment, certain novel compositions are provided which maybe administered alone against, for example, a sensitive or resistantstrain of gram positive bacteria such as S. aureus and E. faecalis.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention represents a unique methodology by which toovercome the increasing resistance of many different varieties of cellsand microorganisms to the antibiotic activity of tetracyclines, theiranalogues and derivatives. The present invention takes into account andacts upon the existence of specific DNA sequences, which are typicallyfound on plasmids and transposons, and which specify proteins fortetracycline-resistance determinants. Some of these determinants act viaan active efflux system which maintains an intracellular tetracyclineconcentration below those levels able to inhibit protein synthesiswithin the microorganism such as described in above-mentioned U.S. Pat.No. 4,806,529. Other determinants act by protecting the ribosome fromtetracycline's inhibitory activity, e.g. by binding with tetracycline.The present invention represents improvement in efficacious and reliabletechniques for overcoming tetracycline resistance in living cells andthus for reestablishing tetracyclines as an antibiotic of choice in thetreatment of infectious diseases caused by the ever-increasing varietyand diversity of disease agents. The invention relies on the action of ablocking agent which is capable of interacting with a product of atleast one tetracycline resistance determinant which acts by protectingthe cell from tetracycline's inhibitory activity. The determinant iscapable of making a product, such as a cytoplasmic protein, whichinteracts with the ribosomes to make them tetracycline resistant or amembrane protein which keeps tetracycline out of the cell.

The present invention is intended for use with tetracycline-resistantcells or organisms which are found to contain or carry a product of thegenetic determinants responsible for tetracycline resistance, and inparticular, those which are due to protection of the ribosome from theinhibitory activity of tetracycline. As described within the recentpublication of Levy, S. B., Journal of Antimicrobial Chemotherapy 24:1-3(1989), the text of which is expressly incorporated by reference herein,more than a dozen different distinguishable tetracycline resistancedeterminants have been uncovered Levy, S. B., "Resistance to theTetracyclines," in Antimicrobial Drug Resistance, (Bryan, L. E.,editor), Academic Press, Orlando, Fla., 1984, pages 191-204; Levy, S.B., ASM News 54:418-421 (1988)!. As these genetic determinants of thesetetracycline-resistant cells have been elucidated, it has becomegenerally accepted that the same or very similar genes are responsiblefor resistance in a large number of different aerobic and anaerobicmicroorganisms.

The present invention is therefore believed suitable for use with atleast, but not exclusively, the following genera: Gram-negative genera,in particular Enterobacteriaceae, which harbor Class A-E tetracyclineresistance determinants; Gram-positive genera including streptococci,staphylococci, and bacillus species which bear the Class K and Ltetracycline resistance determinants; aerobic and anaerobicmicroorganisms bearing the Class M, O or Q determinants represented byStreptococcus agalactiae, Bacteroides, Enterococcus, Gardnerella andNeisseria species, Mycoplasma and Ureaplasma, and Clostridium;Clostridium perfringens bearing the Class P tetracycline-resistantdeterminant.

It will be recognized and appreciated that the above listed organismsare themselves only representative and illustrative of the range,variety, and diversity of cell types, bacterial species, fungi,parasites, and rickettsial disease agents which may be therapeuticallytreated using the present methodology. It will be expressly noted thatno specific class, genus, species, or family of cell, microorganism, orparasite is excluded; to the contrary, it is expected that with futureinvestigations into the determinants responsible for tetracyclineresistance, ever greater numbers of different cells will be recognizedas suitable for efficacious treatment using the present invention. Inaddition, in view of the recent use of tetracyclines for treatment ofneoplasms, it is deemed that the present methodology would be useful insuch therapies van der Bozert et al., Cancer Res. 48:6686-6690 (1988)!.

The present invention represents a major improvement over presentlyknown methods for dealing with tetracycline resistance withindisease-causing cells and organisms. In one preferred embodiment, themethodology requires only two essential steps: the administration to thetetracycline-resistant cell of a predetermined quantity of at least afirst composition selected from the chemical group consisting of ablocking agent which is capable of interacting with a product of atleast one tetracycline resistance determinant which is capable ofprotecting ribosomes in the cell from tetracycline's inhibitoryactivity; and

concomitantly administering to the cell a pre-determined quantity of atleast a second composition selected from the chemical group consistingof tetracycline, tetracycline analogues, and tetracycline derivativeswhich are not said blocking agent.

As noted above, in another embodiment, certain novel compositions areprovided which may be administered alone against, for example, asensitive or resistant strain of gram positive bacteria such as S.aureus and E. faecalis.

Examples of products of a tetracycline resistance determinant are Tet M,Tet O and Tet Q proteins for cytoplasmic protein products and Tet A, TetB, Tet K and Tet L for membrane products.

The resistance mechanism of the cell is allowed to preferentially reactwith the blocking agent so avoiding preferential reaction with thesecond administered composition which is the tetracycline, atetracycline analogue or derivative composition.

Clearly, therefore, it is recognized and understood that in thispreferred embodiment two different compositions are to be administeredconcurrently, sequentially or simultaneously to thetetracycline-resistant cell. Moreover, it will be noted that themethodology requires and relies upon a preferential binding and reactionwith the administered blocking agent in-situ; and consequentlydemonstrate a substantial lack of attraction or preference for the otheradministered tetracycline composition, analogue, or derivative presentin-situ. The operation, utility, and efficacy of the present methodologyis thus based upon an empirically demonstrable preference of thetetracycline-resistant cell for one class of composition over anotherwhen both classes of composition are introduced concomitantly--that is,concurrently, sequentially or simultaneously to the resistant cell.

To date, there is no basis, system, or technique which can be employedto accurately predict which of two similar tetracycline formulations andchemical structures would be preferentially reactive with the resistancesystems of cells. Earlier investigations as described within U.S. Pat.No. 4,806,529 issued Feb. 21, 1989, have demonstrated that whentetracycline i.e.,4-(Dimethylamino)-1,4,4a,5,5a,6-11,12a-octahydro-3,6,10,12,12a-pentahydroxy-6-methyl-1-11-dioxo-2-naphthacenecarboxamide! is administered concurrently or simultaneously withother tetracycline analogues and derivatives such as minocycline orthiatetracycline, it is not actively effluxed from the cell andconsequently enters tetracycline-resistant cells. Further studies havedemonstrated that 13-thiol derivatives of methacycline are able to blockthe efflux protein and inhibit the resistance mechanism in both gramnegative and gram positive cells, including different mechanisms ofresistance, namely efflux and ribosome protection (See U.S. Pat. No.5,064,821). The present invention expands upon these earlierinvestigations in substantial degree. It also provides the user withnovel blocking agents which unexpectedly have been found to show veryhigh inhibition of the mechanisms for ribosome protection as well asefflux.

In one embodiment of the invention, the blocking agent is a tetracyclineanalogue which contains a sufficient part of tetracycline to interactwith a product of at least one tetracycline resistance determinantcapable of protecting cells from tetracycline's inhibitory activity.

One specific class of blocking agents is the class of 13-(substitutedmercapto) tetracyclines of the formula (Formula I): ##STR2## wherein Ais selected from the group consisting of hydrogen and a hydroxyl group;

B is selected from the group consisting of a hydrogen atom, a methylenegroup, and any linear, branched, or ring structure comprising from 1-6carbon atoms and optionally including heteroatoms such as oxygen andnitrogen atoms; and

R is selected from the group consisting of organic entities comprisingfrom 1-12 carbon atoms, with or without other heteroatoms includingsulfur, oxygen, halogen, nitrogen, and the like, and takes form aslinear, branched, or cyclic alkyl, aryl, or alkylaryl structures.

These 13-(substituted mercapto) tetracyclines are known in the art astetracyclines possessing antimicrobial activity in and of themselvesagainst a variety of gram-positive bacteria. This class oftetracyclines, its conventionally recognized pharmacological activity,and methods for its synthesis are described within U.S. Pat. No.3,165,531, the text of which is expressly incorporated by referenceherein.

In one embodiment, the preferred compositions, as empiricallydemonstrated hereinafter, are S-substituted alkyl derivatives at the No.13 carbon ranging from 1-10 carbon atoms in length. Nevertheless, it isexpected that a wide variety of RCO, RCX where X is a halogen, RHC₂, andNRHC₂ analogue and derivative forms in linear, branched, or cyclicstructural format would be useful and operative in the presentmethodology in varying degrees. Accordingly, all such embodiments aredeemed to be within the scope of Formulation I above.

As representative examples of the preferred embodiments of this classwhich were empirically evaluated, some preferred 6-deoxy-13-(substitutedmercapto) tetracyclines (hereinafter "13-S-Derivatives") and theirrespective blocking activities are provided within Table II below. TheK_(j) represents the relative inhibitory activity of each compound. Thelower the K_(j), the more activity against the efflux protein. Thepreferred compound will have a lower K_(j) than tetracycline (i.e.,lower than about 4-8 μg/ml).

                  TABLE II                                                        ______________________________________                                        BLOCKING ACTIVITY OF 13-S-DERIVATIVES OF                                      METHACYCLINE                                                                                 Number of                                                      13-S-Derivatives                                                                             Carbon Atoms                                                                             K.sub.i (μg/ml).sup.1                            ______________________________________                                        Decyl          10         8.0                                                 Hexyl          6          3.1                                                 Cyclohexyl     6          0.4                                                 Benzyl         7          0.9                                                 p-Cl-Benzyl    7          1.5                                                 p-Me-Benzyl    8          1.2                                                 Cyclopentyl,   5          0.5                                                 2-morpholinomethyl                                                            Cyclopentyl    5          0.2                                                 Butyl          4          0.5                                                 t-Butyl        4          0.3                                                 Isobutyl       4          0.1                                                 Propyl         3          0.4                                                 Isopropyl      3          0.4                                                 Dihydroxypropyl                                                                              3          3.9                                                 Ethyl          2          0.4                                                 ______________________________________                                         .sup.1 By everted membrane vesicle assay.                                

From this representative listing, it will be noted that the shorterchain length substitutions or smaller adducts (cyclohexyl vs. hexyl;isobutyl vs. butyl; benzyl vs. parachlorobenzyl) are preferredinhibitors of the efflux system. Also, substitutions at the C2 positionhave only a small effect on the blocking activity. These results lead toa general conclusion that the activity of compositions havingsubstitutions at the 13th carbon relate more to the size of the moleculethan to the charge despite the presence of the sulfur atom. The longerchain length substitutions at the 13th carbon atom (e.g., decyl andhexyl) are not as active as the shorter length substitutions (e.g.,butyl, propyl, and ethyl). Furthermore, the dihydroxypropyl derivativebehaves more poorly in the blocking assay than the propyl or isopropylderivative forms. On this basis, therefore, it is expected that a mostpreferred composition would be one having mercapto-substitutions on the13th carbon atom in which the elipsoidal volume of the substituentjoined to the sulfur atom is in the approximate size range of thatprovided by the butyl, benzyl or cyclopentyl derivatized structures.

Moreover, the data of Table II suggest that the administration to aresistant cell of a 13-substituted mercaptan derivative or a compositionwhich appears structurally similar to a 13-substituted mercaptanderivative would effectively block the resistance mechanism of the cell;and allow the concomitant administration of another tetracycline,tetracycline analogue, or tetracycline derivative to effectively inhibitfurther cell growth.

In another embodiment, the blocking agent which can be employed inpracticing the present invention is the class of C5 esters oftetracyclines of the formula (Formula II): ##STR3## wherein R₁ and R₂are selected from the group consisting of a methylene group, hydroxyl,hydrogen or a group consisting of organic entities comprising from 1-12carbon atoms, with or without other heteroatoms including sulfur,oxygen, halogen, nitrogen, and the like, and takes form as linear,branched, or cyclic alkyl, aryl, or alkylaryl structures; and A isselected from the group consisting of a hydrogen atom, a methylenegroup, and any linear, branched, or ring structure comprising from 1-6carbon atoms and optionally including heteroatoms such as oxygen andnitrogen atoms. Certain C5 esters have been described by Bernardi et al.(Il Farmaco, Ed. Sc. vol. 29--fasc. 12, pages 902-909(1974)) as beinguseful against, for example. S. aureus. Methods of synthesizing thesedisclosed derivatives may be found, for example, in U.S. Pat. No.3,579,564, the disclosure of which is incorporated by reference herein.

In yet another embodiment, hybrids of the above-described6-deoxy-13(substituted mercapto) and C5 ester may be employed as theblocking agent against resistant gram negative strains or alone againstresistant gram positive strains.

In general, the synthesis of these13-thio-substituted-5-acy-6-deoxy-tetracyclines (hereinafter "13,5derivatives) may be accomplished by the anti-Markovnikov radicaladdition of alkyl or aryl thiols to the 6,13 exocyclic double bond ofmethacycline by the method of Blackwood et al., J. Am. Chem. Soc.,85:3943 (1963) the disclosure of which is incorporated by referenceherein, followed by esterification with an appropriate carboxylic acidin anhydrous HF according to the method of Bernardi et al., Il FarmacoEd. Sc., 29:9022 (1974) the disclosure of which is incorporated byreference herein, as depicted in Scheme I below. ##STR4##

FORMULATION, STRUCTURE, AND RANGE OF OTHER TETRACYCLINES, TETRACYCLINEANALOGUES, AND TETRACYCLINE DERIVATIVE FORMS

The present invention requires that at least one other composition whichis not chemically a blocking agent, such as the above-described6-deoxy-13-(substituted mercapto)tetracycline or C5 ester, beadministered concurrently or simultaneously with the blocking agent tothe cell. This additional administered composition is any"tetracycline-type antibiotic" currently known which includestetracycline itself; or any member of the tetracycline family includingall analogues and derivatives which are NOT C5 ester derivatives nor13-carbon substituted mercaptan compounds. Accordingly, the broadestdefinition for the additional tetracycline, analogue, or derivative tobe administered concurrently is defined by Formula III below. ##STR5##wherein R₁ -R₅ may be a hydrogen atom, a halogen atom, a hydroxyl group,or any other organic composition comprising from 1-8 carbon atoms andoptionally include a heteroatom such as nitrogen, oxygen, in linear,branched, or cyclic structural formats. A very wide range and diversityof embodiments within the definition of Formula III as are describedwithin Essentials of Medicinal Chemistry, John Wiley and Sons, Inc.,1976, pages 512-517, the text of which is expressly incorporated byreference herein. Preferably R₁ and R₂ are hydrogen or a hydroxyl group;R₃ is a hydrogen or a methyl group; R₄ is a hydrogen atom, a halogen, ora nitrogen containing entity; and R₅ is a hydrogen atom, or a nitrogencontaining ring structure. The commonly known tetracycline analogues andderivatives including the following: oxytetracycline; chlortetracycline;demeclocycline; doxycycline; chelocardin; minocycline; rolitetracycline;lymecycline; sancycline; methacycline; apicycline; clomocycline;guamecycline; meglucycline; mepyclycline; penimepicycline; pipacycline;etamocycline; and penimocycline. It will be recognized and appreciatedthat these specific tetracycline compositions (as well as many othersconventionally known and available through the scientific literature orfrom commercial sources) may be employed as the alternativetetracycline-type composition which does not contain a C5 ester nor a13-carbon substituted mercapto group as part of its formulation andchemical structure.

The individual compositions embodying Formula I, 13-S-derivatives, orFormula II, C5 esters, or the 13,5 derivative, and Formula III,alternative tetracycline compounds, can be administered concurrently,sequentially or simultaneously in any appropriate carrier for oral,topical or parenteral administration. It is also possible that the twodiscrete compositions could be linked covalently or otherwise joined toeach other and/or to other ligands. These compositions can be introducedby any means that affects an infectious or disease state caused bytetracycline-resistant microorganisms in humans and/or animals. Thespecific route of administration, the choice of carrying materials, andthe particular means for introducing each composition concomitantly tothe tetracycline-resistant cells are of no major importance orrelevance.

Accordingly, if the 13-S-derivative, the C5 derivative composition orthe 13,5 derivative and the other alternative tetracycline-type compoundare to be applied topically, they can be individually or mutuallyadmixed in a pharmacologically inert topical carrier such as a gel, anointment, a lotion, or a cream. Such topical carriers include water,glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides,fatty acid esters, or mineral oils. Other possible topical carriers areliquid petrolatum, isopropylpalmitate, polyethylene glycol, ethanol 95%,polyoxyethylene monolauriate 5% in water, sodium lauryl sulfate 5% inwater, and the like. In addition, materials such as anti-oxidants,humectants, viscosity stabilizers, and the like may also be added if andwhen necessary.

Similarly, if the 13-S-derivative, the C5 derivative composition or the13,5 derivative and the alternative tetracycline-type composition are tobe introduced concurrently, sequentially or simultaneously in parenteralform, each composition will be prepared individually or in combinationin sterile form; in multiple or single dose formats; and be dispersed ina fluid carrier such as sterile physiological saline or 5% dextrosesolutions commonly used with injectables.

Furthermore, if the present methodology is to be employed for oraladministration, each of the two requisite compositions may be providedindividually or in combination in the form of prepared capsules,cachets, or tablets each containing a predetermined quantity of the13-S-derivative, the C5 ester composition or the 13,5 derivative and thetetracycline-type antibiotic. Their preparation may also take form as apowder or granules; or dissolved or suspended in a solution orsuspension within an oil-in-water emulsion or conversely within awater-in-oil liquid emulsion for ingestion or for oral cavity lavagetreatments. These solid or liquid formulations may generally include oneor more carrier materials such as flavoring agents, binders, buffers,diluents, surface active agents, thickeners, lubricants, preservatives,and the like. It is deemed that all of these methods for formulating,preparing, and administering the requisite compositions areconventionally known.

The effective dosages to be employed in vivo are typically dictated bythe intended application or use circumstances; and are generally decidedby reconciling several different factors. First, it will be recognizedand appreciated that each embodiment of the 13-S-derivative, the C5derivative composition or the 13,5 derivative and each embodiment of thealternative tetracycline composition (analogue or derivative) will haveindividual specific pharmacological activity which can be representedand evaluated as the Minimal Inhibitory Concentration (hereinafter"MIC") and as the Minimal Lethal Concentration (hereinafter "MLC")--eachof which varies with its specific formulation and chemical structure.Second, any given specific chemical formulation will also have varyingMIC and MLC dosages which fluctuate with the cell type--as, for example,with the genus and species of microorganism; thus, the MIC and MLC ofeach individual composition will vary markedly--as, for example, whenadministered to gram-positive bacteria in comparison to gram-negativebacteria or to the various different genera of fungi, rickettsia, andparasites. Thirdly, the degree of tetracycline resistance is known tovery substantially among the different cell types, their delineatedgenera, and among the different species comprising a single genus; thisvarying degree of tetracycline resistance is without regard to whetherthe mechanism of resistance is based upon an active efflux system or aribosome protection system intracellularly. Lastly, each specific routeof in vivo administration is conventionally recognized to requiremarkedly different dose concentration of conventionally knowntetracycline compounds; accordingly, in vivo therapeutic dosages willvary depending upon whether the tetracycline-type composition is givenorally, parenterally, or topically. Each of these individual factorsshould be taken in consideration by the user when deciding the properdosage or concentration for the 13-S-derivative, the C5 derivativecomposition or the 13,5 derivative and the other tetracycline antibioticcomposition.

In general, however, it is most desirable that the dosage andconcentration of the 13-S-derivative, the C5 derivative composition(broadly defined by Formula I or Formula II, respectively) or the 13,5derivative be administered in a subinhibitory quantity--that is, lessthan the minimum inhibitory quantity--that is, less than the minimuminhibitory concentration or the minimum lethal concentration for thatspecific composition when employed against a tetracycline-resistantcell. In comparison, it is essential that the chosen alternativetetracycline-type composition (tetracycline analogue or tetracyclinederivative meeting the broad definitional requirements of Formula IIIabove) be employed in at least a minimum inhibitory concentration; and

preferably be administered at an effective dosage to provide a minimallethal concentration in-situ. Accordingly, it is deemed that theconcentrations for the two concomitantly administered compositions areconventionally known within the art; and can be optimalized with aminimum of difficulty.

Again, as noted above, certain novel compounds, in particular, the 13,5derivatives, have been found to be particularly useful against certaingram positive bacteria when administered alone.

In this context, and as empirically demonstrated by the data of Charts1-5 for the 13-S-derivatives, Charts 6-7 for the C-5 derivatives andCharts 8-9 for the 13,5 derivatives which follow hereinafter, theconcomitant administration of the 13-S-derivative, the C5 derivativecomposition or the 13,5 derivative and the other tetracycline-typecomposition together provides not only means for overcoming tetracyclineresistance but also offers the capability to enhance the pharmacologicalactivity of the known tetracycline-type composition to exert cidalactivity and cidal effects upon the cell. Contrary to the universallyaccepted conventional view that tetracyclines, regardless offormulation, are only bacteriostatic agents--i.e., agents that do notkill but only inhibit future growth, the present method provides asynergistic combination of compositions which enhances the antibioticactivity of the tetracycline-type composition; and, for the first time,allows the enhanced tetracycline-type composition to exert bacteriocidalpowers, "cidal" capability, i.e., the ability to kill the cell ratherthan merely inhibit its growth, against a broad spectrum of bacteria.

In addition, the general molar ratio of 13-S-derivative, the C5derivative composition, or the 13,5 derivative to alternativetetracycline-type composition is expected generally to be from0.01:100.0, and is preferably in the range from 0.05:2.0. It is mostdesirable, however, that in no instance should the dosage of the13-S-derivative, the C5 derivative composition or the 13,5 derivative beemployed in a concentration which is within the MIC or MLC values. Incomparison, the alternative, tetracycline-type composition (tetracyclineor tetracycline analogue or tetracycline derivative) should beadministered in accordance with conventional practice for theefficacious therapeutic treatment of infection or disease in humansand/or animals. Accordingly, for therapeutic purposes, the daily dosageof 13-S-derivative, the C5 derivative composition or the 13,5 derivativefor treatment of disease in living mammals is expected to lie in therange from 0.01-100 mg/kg (preferably from 15 to 30 mg/kg) of normalbody weight while the dosage of the other tetracycline, analogue orderivative should continue to be given in the range from 500 milligramsto 2.0 grams per day depending upon the age, weight, and route ofadministration.

When the 13,5 derivative is administered alone, for example, fortreating infections caused by gram positive bacteria such as S. aureusor E. faecalis, the dosage employed is preferably that used inconventional tetracycline therapy.

It will also be understood that the normal, conventionally known,precautions will be taken regarding the administration of tetracyclinesgenerally in order to ensure their efficacy under normal usecircumstances. Especially when employed for therapeutic treatment ofhumans and animals in vivo, the practitioner should take all sensibleprecautions to avoid conventionally known contradictions and toxiceffects. Thus, the conventionally recognized adverse reactions ofgastrointestinal distress and inflammations, the renal toxicity,hypersensitivity reactions, changes in blood, and impairment ofabsorption through aluminum, calcium, and magnesium ions should be dulyconsidered in the conventional manner.

MODE AND MANNER OF PHARMACOLOGICAL ACTIVITY

It must be emphasized again that the present methodology is useful withall cells, regardless of type, source, family, genus, or species whichhave genetic determinants for tetracycline resistance. The methodologyof the present invention is suitable for use with both tetracyclineresistance attributable to an active efflux transport system utilizingone or more TET proteins which actively bind with tetracycline-typeantibiotics and transport the tetracycline composition out of thecytoplasm of the cell; and also with tetracycline resistance which is anonefflux system and typically involves a ribosome protection mechanismwhich causes a tetracycline antibiotic to fail to inhibit proteinsynthesis intracellularly. Regardless of which tetracycline resistancemechanism is present within the resistant cell, the present methodologyis effective in overcoming tetracycline resistance and in rendering thecell tetracycline-sensitive. Although the sequence of molecularreactions remains far from understood at the present time, theconcurrent or simultaneous administration of at least one13-S-derivative or the C5 derivative composition prepared in accordancewith Formula I or Formula II or the 13,5 derivative and at least oneother tetracycline antibiotic composition in accordance with Formula IIIcauses an in situ conversion of the cell from a resistant state into atetracycline-sensitive state.

The efficacy and utility of the present methodology is based upon thecell's unexpected preferential reaction with the 13-S-derivative, the C5derivative composition or the 13,5 derivative which is desirably presentin a subinhibitory concentration; and the comparable absence of avidityby the cell for the other tetracycline-type composition concomitantlyadministered. The resistance mechanism of the cell--be it the activeefflux system or the ribosome protection system--focuses upon andinteracts with the 13-S-derivative, the C5 derivative composition or the13,5 derivative primarily and predominantly; the concurrent orsimultaneous presence of the other tetracycline-type antibioticcomposition is relatively ignored and effectively overlooked by thetetracycline-resistance mechanism of the cell.

Consequently, the other tetracycline-type antibiotic composition isallowed to accumulate intracellularly in at least a minimum inhibitoryconcentration (and preferably in a minimum lethal concentration); andthis other tetracycline-type antibiotic is able to bind to the ribosomesand to exert its recognized pharmacological activity intracellularly toprevent further protein synthesis within that cell. In this respect, theadministered 13-S-derivative of Formula I or C5 derivative compositionof Formula II or 13,5 derivative is clearly the preferred compositionfor reaction with the tetracycline-resistance mechanism present; and bythis preferred reactivity, acts as a blocking agent to engage and todivert the tetracycline resistance mechanism of that cell to the extentthat the concurrently or simultaneously administered othertetracycline-type antibiotic composition of Formula III is able to exertits characteristic pharmacological activity efficaciously against thecell and to prevent further protein synthesis intracellularly. Thepresent methodology is thus effective and useful by the cell's ownpreference for engagement and reaction with the 13-S-derivative, the C5derivative composition or the 13,5 derivative, rather than with theconcomitantly administered other tetracycline-type antibiotic. In thismanner, the cumulative effect is to render the celltetracycline-sensitive for therapeutic purposes.

EXPERIMENTAL STUDIES

A series of experiments and resulting empirical data demonstrate andevidence the efficacy and utility of the present invention. Theseexperiments will illustrate the essential components of the presentmethodology, and will demonstrate the value of the preferred embodimentscomprising 13-S- or C5 derivative compositions prepared in accordancewith Formula I, or Formula II and document the range and diversity ofsome tetracycline-resistant microorganisms which can be renderedsensitive to tetracycline therapy by employing the present methodology.

For purposes of conducting the experimental model, tetracycline for the13-S-derivative or doxycycline for the C5 derivative was employeduniformly in combination with a variety of different C5 or 13substituted mercapto-tetracyclines.

Nevertheless, it will be understood that doxycycline and tetracyclineare employed merely as a representative of all the differentcompositions and embodiments of tetracyclines, tetracycline analogues,and tetracycline derivatives conforming to the definition of Formula IIIgiven previously; and that the present invention is not limited to theuse of tetracycline alone as a specific chemical formulation andstructure. Moreover, it will be understood that the experiments andempirical data presented hereinafter are merely illustrative examples ofthe present invention without regard to specific applications orparticular uses; and that the described experiments are merelyrepresentative of the totality of embodiments encompassed within thescope of the present invention.

EXPERIMENTAL SERIES 1

Initially, the inhibitory effects of a variety of different13-S-derivatives in comparison to tetracycline and minocycline wereexamined using a variety of different bacteria. These includedtetracycline sensitive (hereinafter "Tc^(s) ") and tetracyclineresistant (hereinafter "Tc^(r) ") strains of E. coli, S. aureus, and E.faecalis. The general protocol for performing these experiments is asfollows: Cultures were grown up fresh in L broth in the morning from anovernight culture. After 4-6 hours of growth, each bacterial culture wasdiluted to an A₅₃₀ of 0.2-0.5 depending on the strain (E. coli, 0.5; S.aureus, 0.4; E. faecalis, 0.2). Individual tubes, containing 1 ml of Lbroth and different concentrations of 13-S-derivatives, were inoculatedwith the different bacterial cultures and then incubated at 37° C. After17-18 h of incubation, the concentration of each 13-S-derivative atwhich no observed cloudiness was seen was called the minimal inhibitoryconcentration (MIC). The minimal lethal concentration (MLC), i.e., thatconcentration which kills 99.9%, was based on the number of bacteriainitially inoculated into the assay tubes. Those culture tubes showingno bacterial growth after incubation at 37° C. were evaluated for thenumber of bacteria remaining.

The results obtained in this experimental series are provided by TablesE1-E3 below.

                                      TABLE E1                                    __________________________________________________________________________    SUSCEPTIBILITY TESTING OF E. coli                                                               Class A Tc.sup.r                                                                         Class B Tc.sup.r                                 Tc.sup.s (ML308-225)                                                                            (D1-299)   (D1-209)                                         Drug  MIC (μg)                                                                         MLC (μg)                                                                         MIC (μg)                                                                         MLC (μg)                                                                        MIC (μg)                                                                         MLC (μg)                                __________________________________________________________________________    Tetracycline                                                                         0.6 ± 0.26                                                                        40 3≡ 0.26                                                                   160 ± 0.25                                                                      200  >200  >200                                       Minocycline                                                                         <4    <4    <4     20  6 ± 2                                                                            30 ± 10                                 Benzyl*                                                                             16 ± 6                                                                           25 ± 6                                                                           46 ± 20                                                                          160 ± 50                                                                          30 ± 0.26                                                                      60 ± 10                                 Cyclohexyl*                                                                         60 ± 10                                                                          120    100 ± 0.26                                                                      >200 ND    ND                                         Cyclopentyl*                                                                        20     60     40 ± 0.26                                                                      80   80 ± 40                                                                          100 ± 60                                Propyl*                                                                             30 ± 6                                                                           60 ± 10                                                                            40 ± 0.26                                                                      60   60    80                                         Isopropyl*                                                                          22 ± 2                                                                           60    46 ± 6                                                                           60   35 ± 6                                                                           45 ± 16                                 Ethyl*                                                                                6 ± 0.26                                                                       60    14 ± 4                                                                           46 ± 6                                                                          35 ± 16                                                                          60 ± 10                                 __________________________________________________________________________     *Note: ± 0.25 indicates that the same MIC or MLC was determined in two     or more experiments. Other values represent experimental error determined     by averaging the values obtained in multiple experiments. If no value is      given, the experiment has not be repeated. Larger numbers will                consistently have larger errors since all experiments were done by the        standard 1 ml serial dilution liquid MIC procedure.                           ND = not done.                                                                Tc.sup.s = tetracycline sensitive strain.                                     Tc.sup.4 = tetracycline resistant strain.                                     * = Smercapto derivative of methacycline.                                

                  TABLE E2                                                        ______________________________________                                        SUSCEPTIBILITY TESTING OF S. aureus                                                  Tc.sup.s (RN450)                                                                            Tc.sup.r (RN4250)                                        Drug     MIC (μg)                                                                             MLC (μg)                                                                             MIC (μg)                                                                           MLC (μg)                              ______________________________________                                        Tetracycline                                                                           0.75 ± 0.25                                                                          >6        90 ± 10                                                                             100                                     Minocycline                                                                            <0.25     8         <0.25   >80                                      Benzyl   0.2 ± 0.1                                                                            10 ± 2   1 ± 0.25                                                                         10 ± 4                                Cyclohexyl                                                                              2.5 ± 1.25                                                                          10 ± 5 1.5 ± 0.5                                                                          10 ± 4                                Cyclopentyl                                                                            1         5           2 ± 0.25                                                                          6 ± 2                                Propyl   0.5 ± 0.25                                                                           5           4 ± 0.25                                                                         16 ± 4                                Isopropyl                                                                              0.5 ± 0.25                                                                            6 ± 2 4.5 ± 0.5                                                                          8                                        Ethyl    0.5 ± 0.25                                                                           4         5 ± 2                                                                               30 ± 10                              ______________________________________                                         Tc.sup.s = tetracycline sensitive strain                                      Tc.sup.r = tetracycline resistant strain                                 

                                      TABLE E3                                    __________________________________________________________________________    SUSCEPTIBILITY TESTING OF E. faecalis                                                           Tc.sup.r (L)                                                                              Tc.sup.r (M)                                    Tc.sup.s (ATCC9790r)                                                                            ATCC9790r/TetL)                                                                           (ATCC09790r/TetM)                               Drug  MIC (μg)                                                                         MLC (μg)                                                                         MIC (μg)                                                                         MLC (μg)                                                                         MIC (μg)                                                                        MLC (μg)                                __________________________________________________________________________    Tetracycline                                                                         0.26 >200  90 ± 10                                                                          >300  100  300                                        Minocycline                                                                         <0.26  >40  <0.26  >80   10  >80                                        Benzyl*                                                                              0.6 ± 0.25                                                                        8 ± 0.26                                                                       0.76 ± 0.26                                                                      8 ± 2                                                                            3.6 ± 1                                                                         18 ± 2                                  Cyclohexyl*                                                                         1.26 ± 0.26                                                                      8     1.5 ± 0.5                                                                        8 ± 2                                                                            2.6 ± 0.6                                                                         10 ± 0.25                             Cyclopentyl*                                                                          1 ± 0.26                                                                       10 ± 4                                                                             1 ± 0.5                                                                        18 ± 2                                                                           3 ± 1                                                                           >16                                        Propyl*                                                                              1.6 ± 0.26                                                                        40 ± 0.25                                                                      2.6 ± 0.6                                                                        60 ± 10                                                                          16 ± 2                                                                          30 ± 10                                 Isopropyl*                                                                          3 ± 1                                                                              20 ± 0.26                                                                      3 ± 1                                                                             100 ± 0.25                                                                      22 ± 2                                                                          >200                                       Ethyl*                                                                                1 ± 0.6                                                                          40 ± 0.25                                                                      4 ± 1                                                                             100 ± 0.25                                                                      25 ± 6                                                                          >200                                       __________________________________________________________________________     Tc.sup.s = tetracycline senstive strain.                                      Tc.sup.r = tetracycline resistant strain.                                

A close inspection and reading of Tables E1-E3 will reveal the followingpoints regarding the tetracycline susceptible strains and thetetracycline resistant strains tested. These are:

Susceptible Strains

1. E. coli (Table E1. Column 1)

None of these compounds was more active than tetracycline or minocyclineagainst susceptible E. coli strains. The most active was theethyl-S-derivative which showed an MIC of 5 μg/ml.

2. S. aureus (Table E2, Column 1)

Against susceptible S. aureus, all of the 13-S-derivatives wereeffective alone within therapeutic ranges. They were about as active astetracycline and minocycline (except perhaps the cyclohexyl derivative).All 13-S-derivatives showed bacteriocidal activity better thantetracycline or minocycline of which 4 showed bacteriocidal activity ata level of about 5 μg/ml. 3. E. faecalis (Table E3, Column 1)

Against susceptible Enterococcus faecalis, all the tested compositionswere effective well within a therapeutic range and all, but theisopropyl derivative, at 1 μg/ml or less. All showed greaterbacteriocidal activity than did tetracycline or minocycline, especiallythe benzyl, cyclohexyl, and cyclopentyl S-derivatives.

Resistant Strains

1. All the other compositions were more active than tetracycline againstresistant E. coli strains (both Class A and Class B determinants). Noneindividually was as active as minocycline. Most 13-S-derivatives showedbacteriocidal activity lower than tetracycline against resistant E.coli, but not within therapeutic ranges (Table E1, Columns 2 and 3).

2. Against resistant S. aureus, all the tested compounds showed an MICwithin a therapeutic range, at least 20-100 fold more active thantetracycline. None individually was as active as minocycline. All13-S-derivatives were more bacteriocidal than tetracycline orminocycline alone with cyclopentyl showing an MLC of 6±2 μg/ml. Benzyl,cyclohexyl, and cyclopentyl S-derivatives each showed similar MIC valuesand MLC values against susceptible and resistant S. aureus. The mostactive 13-S-derivative was the cyclopentyl form (Table E2, Column 2).

3. All the tested compositions had an MIC within a therapeutic rangeagainst E. faecalis bearing the Tet L determinant:benzyl>cyclopentyl>cyclohexyl, followed by the others. The13-S-derivatives were equally effective by MIC against susceptible andTet L containing Enterococcus. All were more bacteriocidal thantetracycline and minocycline individually; the MLC for benzyl andcyclohexyl was 8±2 μg/ml (Table E3, Column 2).

4. Against Tet M containing E. faecalis, all the other tested compoundswere considerably more antibacterial than tetracycline. Three of them,the benzyl, cyclohexyl, and cyclopentyl derivatives also had MIC valuesbelow minocycline and within therapeutic levels (Table E3, Column 3).Bacteriocidal activity was observed, but above therapeutic levels.

5. While the MLC against resistant S. aureus and E. faecalis was 8-10μg/ml for the most active drugs, a killing effect (seen as a 10-99% dropin cell viability) by the analogues occurred at considerably lower drugconcentrations (see charts).

EXPERIMENTAL SERIES 2

Subsequently, another series of experiments was conducted which employedconcurrent administrations of tetracycline and at least one other13-S-derivative composition in accordance with Formula I above. Thegeneral experimental protocol for synergy studies followed substantiallythat procedure employed for the standard MIC and MLC assays. Theorganisms were grown in fresh L broth and inoculated in culture tubescontaining different concentrations of 13-S-derivative compositions andtetracycline together. The previously described methods for determiningMIC and MLC were otherwise followed.

Accordingly, the results are provided by Charts 1-5 in which: Chart 1represents the concurrent administration of 13-cyclopentyl sulfidederivative of methacycline in varying proportional ratios totetracycline; Chart 2 represents 13-propyl-sulfide derivatives ofmethacycline varying proportional ratios with tetracycline; Chart 3represents varying proportional ratios with tetracycline; Chart 3represents varying proportional ratios of 13-cyclohexyl-sulfidederivatives of methacycline and tetracycline administered concurrently;Chart 4 represents varying proportional ratios of 13-benzyl-sulfidederivatives of methacycline delivered concurrently with tetracycline;and Chart 5 illustrates the concurrent administration of varyingproportions of 13-ethyl-sulfide derivatives of methacycline andtetracycline.

                  CHART 1                                                         ______________________________________                                        MIC/MLC (μg/ml) dosages                                                    for tetracycline-resistant strains                                            using cyclopentyl-sulfide derivatives of                                      methacycline with and without tetracycline                                    ______________________________________                                         ##STR6##                                                                      ##STR7##                                                                      ##STR8##                                                                      ##STR9##                                                                     ______________________________________                                         ##STR10##                                                                

    CHART 2                                                                       ______________________________________                                        MIC/MLC (μm/ml) dosages                                                    for tetracycline-resistant strains                                            using propyl-sulfide derivatives of                                           methacycline with and without tetracycline                                    ______________________________________                                         ##STR11##                                                                     ##STR12##                                                                     ##STR13##                                                                     ##STR14##                                                                    ______________________________________                                         ##STR15##                                                                

    CHART 3                                                                       ______________________________________                                        MIC/MLC (μg/ml) dosages                                                    for tetracycline-resistant strains                                            using cyclohexyl-sulfide derivatives of                                       methacycline with and without tetracycline                                    ______________________________________                                         ##STR16##                                                                     ##STR17##                                                                     ##STR18##                                                                     ##STR19##                                                                    ______________________________________                                         ##STR20##                                                                

    CHART 4                                                                       ______________________________________                                        MIC/MLC (μg/ml) dosages                                                    for tetracycline-resistant strains                                            using benzyl-sulfide derivatives of                                           methacycline with and without tetracycline                                    ______________________________________                                         ##STR21##                                                                     ##STR22##                                                                     ##STR23##                                                                     ##STR24##                                                                    ______________________________________                                         ##STR25##                                                                

    CHART 5                                                                       ______________________________________                                        MIC/MLC (μg/ml) dosages                                                    for tetracycline-resistant strains                                            using ethyl-sulfide derivatives of                                            methacycline with and without tetracycline                                    ______________________________________                                         ##STR26##                                                                     ##STR27##                                                                     ##STR28##                                                                     ##STR29##                                                                    ______________________________________                                         ##STR30##                                                                      As evidenced by the data of Charts 1-5, the results of administering        13-S-derivative tetracycline compositions concurrent with varying             proportional ratios of tetracycline clearly support the following         

1. Against the tetracycline resistant (Class A) E. coli (strain D1-299)synergy was observed. The most effective analogues were cyclopentyl,cyclohexyl, and ethyl. These all inhibited growth at concentrations of 5μg/ml or less of analogue and tetracycline. Synergy was alsodemonstrated in bacteriocidal activity, although the amounts of the13-S-derivatives needed were higher than 5 μg/ml in order to kill 99.9%of the cells with 4-5 μg/ml of tetracycline.

2. Against tetracycline resistant S. aureus, all the 13-S-derivativestested showed synergistic activity at levels of both drugs below 4μg/ml. In addition, cyclohexyl>cyclopentyl>benzyl showed bacteriocidalactivity within therapeutic combinations with tetracycline where thecombined dose of the two drugs was≦6 μg/ml to achieve MLC.

3. Against E. faecalis (Tet L), all four 13-S-derivatives showedexcellent synergy in inhibiting growth in combination: <1 μg/ml ofanalogue with 1 μg/ml tetracycline. While bacteriocidal effects wereseen synergistically, the amounts of drugs needed to produce the MLCwere higher than each at 4-5 μg/ml.

4. Against E. faecalis (Class M) cyclopentyl, cyclohexyl, and benzylS-derivatives showed little, if any synergistic activity withtetracycline. However, the propyl-S-derivative, while not as activealone, did show meaningful synergy.

SUMMARY

1. These studies show that a group of S-alkyl substitutions and thebenzyl substitution at the 13th carbon position of methacycline caninhibit growth of both susceptible and tetracycline resistantgram-positive (and to a less extent gram-negative) organisms.

2. In combination with tetracycline, all of these 13-S-derivatives showsynergy, both in growth inhibition and in bacteriocidal activity forgram-positive as well as gram-negative susceptible and resistantstrains.

3. All of the 13-S-derivatives tested show bacteriocidal activity,although this is most evident against the gram-positive bacteria testedalone and in synergy with tetracycline, and against E. coli in synergywith tetracycline. 4. All the 13-S-derivatives tested alone show greaterbacteriocidal activity than minocycline against S. aureus and E.faecalis, chiefly the benzyl, cyclohexyl, and cyclopentyl derivatives.

EXPERIMENTAL SERIES 3

Everted vesicles, to which the TET proteins responsible for tetracyclineefflux are attached, provide a reliable method for measuring efflux fromtetracycline-resistant bacteria. By exposing the vesicles to differentconcentrations of tetracycline and measuring the amount of tetracyclinetaken up by the vesicle, the affinity of tetracycline for the effluxsystem may be determined. Similarly, exposure of the vesicles tosolutions having both tetracycline and a potential efflux proteinblocking agent produces a competition between tetracycline and theblocking agent for the limited number of binding states on the TETproteins. Subsequent measurement of the tetracycline concentrationwithin the everted vesicles thus provides a sound measurement of thefunction of the bacterial efflux system for tetracycline in the presenceof the potential blocking agent. This assay has also identified agentswhich affect resistance specified by other mechanisms, namely by acytoplasmic protein which protects ribosomes from the inhibition oftetracycline (see U.S. Pat. No. 5,064,821).

In the experiments, 0.5 mg/ml of everted membrane vesicles of E. colistrain D1-209 bearing the Class B tetracycline resistance determinantwere incubated with about 4 μM of ³ H-tetracycline in a volume of 300μl. Different potential blocking agents with substitutions at the C5position were separately tested at concentrations of 0.2, 0.5 and 2μg/ml. A control experiment, wherein no blocking agent was used, wasalso performed. After incubation for 2.5 minutes, the vesicles werecollected on membrane filters and the effect of the blocking agent onuptake of ³ H-tetracycline was assessed by liquid scintillation countingof the radioactivity on the filters.

The assay showed the relative inhibition of tetracycline by thedifferent drugs vis a vis drug amounts (Table E4 below). Using theuptake at 2.5 minutes (when the system reaches equilibrium) the IC₅₀ ofthe analogs was determined. Using this method, the IC₅₀ of different C5esters ranged from 0.2 μM (5 proprionate methacycline) to 9.4 μM (5cyclopropanoate methacycline). Some showed no effect in this assay,suggesting they have poor, if any, blocking activity. These studiessuggested that the smaller substitution at the 5 position, e.g., theproprionates and phenyl acyl, were more effective blockers of the effluxsystem than were those with larger substitutions. Derivatives bearing asubstitution at C13 and C5 were also effective (e.g.,13-cyclopentyl-thio-5-proprionate tetracycline, IC₅₀ =3.3 μM). Some ofthe drugs from this assay were then tested for their activity againstthe whole bacterial cells (See Experimental Series 4).

                  TABLE E4                                                        ______________________________________                                        5-Esters                                                                       ##STR31##                                                                                                        IC.sub.50                                 Cmpd   R               R.sub.1      (μM).sup.a                             ______________________________________                                        1      CH.sub.3, H     COCH.sub.2 CH.sub.3                                                                        1.0                                       2      CH.sub.2        COCH.sub.2 CH.sub.3                                                                        0.2                                       3      CH.sub.3, H     COCH.sub.2 C.sub.6 H.sub.5                                                                 1.6                                       4      CH.sub.3, H     CO(CH.sub.2).sub.6 CH.sub.3                                                                9.3                                       5      CH.sub.2 S-cyclopentyl, H                                                                     COCH.sub.2 CH.sub.3                                                                        3.3                                       6      CH.sub.3, H     COCH.sub.2 CH.sub.2 CO.sub.2 H                                                             2.0                                       7      CH.sub.3, H     (CH.sub.2).sub.3 NH.sub.2                                                                  6.4                                       8      CH.sub.2 S-propyl                                                                             COCH.sub.2 CH.sub.3                                                                        1.4                                       9      CH.sub.2 S-cyclopentyl, H                                                                     cyclohexanoate                                                                              NE*                                      10     CH.sub.2 S-cyclopentyl, H                                                                     cyclopentanoate                                                                             NE*                                      11     CH.sub.2        cyclopropanoate                                                                            9.4                                       ______________________________________                                         .sup.a by everted vessicle assay                                              *NE = no effect (>30 μM)                                              

EXPERIMENTAL SERIES 4

The growth inhibitory effect of different C5 derivatives oftetracyclines, with and without doxycycline, were determined usingsensitive and resistant E. coli, Staphylococcus aureus, and Enterococcusfaecalis. The general protocol for these experiments was as follows:

Cultures were grown up fresh in L broth in the morning from an overnightculture. After 4-6 hours of growth, each bacterial culture was dilutedto approximately 5×10⁵ cells/ml. The drugs were diluted in two-folddilutions from 50 μg/ml to <1 μg/ml and tested alone and in mixtures byincubation for 18 h at 37° C. The MIC was that concentration of drugalone or combination of drugs in which no growth (no cloudiness) wasobserved. The minimal lethal concentration (MLC) was that concentrationwhich killed 99.9% of the cells and was based on the number of bacteriainitially inoculated into the-assays. Those cultures showing nobacterial growth after incubation at 37° C. were evaluated for thenumber of viable bacteria remaining by plating onto nutrient agarplates; these data determined the MLC. The results of four prototypedrugs, the C5 propyl ester of methacycline, the C5 propyl ester ofdoxycycline and the combination of C5 propyl, C13 derivatives(13-cyclopentyl-thio-5-proprionate tetracycline and13-propyl-thio-5-proprionate tetracycline) are presented in Table E5 andCharts 6-9.

                  TABLE E5                                                        ______________________________________                                        Susceptibility of Tetracycline Susceptible Strains (MIC, μg/ml)                          E. coli   S. aureus                                                                              E. faecalis                                  C5 Ester      ML308     450      ATCC 9790                                    ______________________________________                                        5 proprionate methacycline                                                                  10        1.3      .8                                           5 proprionate doxycycline                                                                   10        .6       .4                                           13-cyclopentyl-thio-5-                                                                      20        .4       .4                                           proprionate tetracycline                                                      13-propyl-thio-5-                                                                           20        .3       2.5                                          proprionate tetracycline                                                      ______________________________________                                    

Summary of Charts 6-9

Chart 6 MIC/MLC (μg/ml) dosages for tetracycline resistant strains using5 proprionate methacycline with and without doxycycline.

Chart 7 MIC/MLC (μg/ml) dosages for tetracycline resistant strains using5 proprionate doxycycline with and without doxycycline.

Chart 8 MIC/MLC (μg/ml) dosages for tetracycline resistant strains using13-cyclopentyl-thio-5-proprionate tetracycline with and withoutdoxycycline.

Chart 9 MIC/MLC (μg/ml) dosages for tetracycline resistant strains using13-propyl-thio-5-proprionate tetracycline with and without doxycycline.

+=growth

0=no growth (MIC)

↓=killing

▪=99.9% killing (MLC)

□=no growth, but no microbiologic testing

The analog concentration is given in columns A and H. It is thisconcentration which is within the squares 2-10. The deoxycyclineconcentration in the control is in Column 11 and its concentration ineach of the boxes is given as small numbers within each of the squares.

                                      CHART 6 A, B                                __________________________________________________________________________     ##STR32##                                                                     ##STR33##                                                                    __________________________________________________________________________

                                      CHART 6 C, D                                __________________________________________________________________________     ##STR34##                                                                     ##STR35##                                                                    __________________________________________________________________________

                                      CHART 7 A, B                                __________________________________________________________________________     ##STR36##                                                                     ##STR37##                                                                    __________________________________________________________________________

                                      CHART 7 C, D                                __________________________________________________________________________     ##STR38##                                                                     ##STR39##                                                                    __________________________________________________________________________

                                      CHART 8 A, B                                __________________________________________________________________________     ##STR40##                                                                     ##STR41##                                                                    __________________________________________________________________________

                                      CHART 8 C, D                                __________________________________________________________________________     ##STR42##                                                                     ##STR43##                                                                    __________________________________________________________________________

                                      CHART 9 A, B                                __________________________________________________________________________     ##STR44##                                                                     ##STR45##                                                                    __________________________________________________________________________

                                      CHART 9 C, D                                __________________________________________________________________________     ##STR46##                                                                     ##STR47##                                                                    __________________________________________________________________________

All four tetracycline derivatives effectively inhibit growth ofsensitive Staph aureus and E. faecalis when used alone. None waseffective alone against susceptible E. coli (Table E5). Against thetetracycline resistant S. aureus and E. faecalis strains, the analogswere also very effective alone (Charts 6-9). While they were noteffective alone against resistant E. coli D1-299, they did showsynergistic effects achieving both decreased growth and cell killing.For instance, at a combination of 5.9 μg/ml of doxycyline and 6.25 μg/mlof 13-cyclopentyl-thio-5-proprionate Tc, there was an MLC of E. coliD1-299 (Chart 8A). The 13-cyclopentyl-thio-5-proprionate tetracyclinewas equally effective alone against.Tet K in S. aureus and Tet L and TetM determinants in E. faecalis. In fact, the values of the MLC/MLC wereclose to those of the sensitive strains (Table E5).

The C5 proprionate esters were also effective alone against resistant S.aureus and E. faecalis, and showed further efficacy in additiveness andsynergy when used with doxycycline in MIC and MLC. For instance, inChart 6B, the combination of 1.55 μg/ml doxycycline and 1.56 μg/ml5-proprionate methacycline or 6.23 μg/ml doxycycline and 0.78 μg/mlanalog achieved MIC.

The growth inhibition included cells bearing different tetracyclineefflux systems (Class A, B, K & L) and a ribosomal protection system(Class M). These unexpected results confirm that the substitution at theC5 position produces an effective efflux blocking agent whichdemonstrates synergistic antibacterial activity against tetracyclineresistant bacteria bearing different resistance determinants.

EXAMPLE 1 Synthesis of 13-propylthio-5-hydroxy-6-α-deoxy-tetracycline

Methacycline hydrochloride (5.0 g, 10.4 mmol) was placed in around-bottom flask and suspended in 100 mL of EtOH. Twenty mL ofpropanethiol (16.8 g, 0.270 mol) and AIBN 250 mg, were added and thereaction mixture refluxed with stirring for 12 h while under N₂. Themixture was reduced to 1/5 volume by distillation and filtered. Thefiltrate was dripped slowly into cold Et₂ O while stirring resulting inthe formation of a yellow precipitate. The precipitate was filtered,dissolved in H₂ O and brought to pH 4.5 with 1.0M NaOH. This solutionwas filtered, and extracted with CH₂ Cl₂ yielding a dark yellow solid(620 mg). The solid was dissolved in MeOH and treated with charcoalyielding a yellow solid in low yield (25%, 256 mg) mp=130°-140° C.(dec.). TLC r_(f) =0.70 (I); HPLC R_(t) =20.18 min. HNMR (DMSO-d₆) δ7.50(t, 1H), 7.05 (d, 1H), 6.85 (d, 1H), 4.32 (d, 2H), 3.15 (s, 1H), 2.65(s, 6H), 2.32-2.52 (m, 2H), 1.51-1.80 (m, 2H), 0.9-1.22 (m, 3H); HRMS(FAB); calc for C₂₅ H₃₀ N₂ O₈ S 519.1801 (M+1), found 519.1815 (M+1).

EXAMPLE 2 Synthesis of13-cyclopentylthio-5-hydroxy-6-α-deoxy-tetracycline

This compound was prepared substantially as described in Example 1.Purification was either by column chromatography on EDTA silica,extraction pH 4.5 into CH₂ Cl₂, or by HPLC chromatography. An analyticalsample was produced by HPLC as a yellow solid of mp=132°-139° C. (dec.)in moderate yield (28.3%). Higher yields were obtained by the extractionmethod and treatment with activated charcoal in MeOH (32.1%); TLC r_(f)=0.80 (I); HPLC R_(t) =21.19 min. HNMR (MeOH-d₄) δ7.38 (t, 1H), 7.02 (d,1H), 6.72 (d, 1H), 4.10 (s, 2H), 2.70 (br s, 6H), 1.81-2.01 (br m, 2H),1.28-1.75 (br m, 6H), (br m, 2H); HRMS (FAB); calc for C₂₇ H₃₂ N₂ O₈ S545.1957 (M+1), found 545.1960 (M+1).

EXAMPLE 3 Synthesis of 13-propylthio-5-proprionate-6-deoxy-tetracycline

100 mg of 13-propylthio-5-hydroxy-6-α-deoxy-tetracycline of Example 1and 2.0 g of propionic acid were dissolved in 20 mL of anhydroushydrofluoric acid and the resultant solution sealed in a polypropylenetube for 3 days at room temperature. The hydrofluoric acid was removedby a slow steady stream of nitrogen and the residue taken up in diethylether. The precipitate was dissolved in MeOH (4 mL) and injected into apreparative HPLC utilizing a C18 reverse-phase column and mobile phasesof phosphate buffer (pH 4.5) and MeOH over a linear gradient (30%-100%over 30 minutes) at 30 mL/minute. The compound was collected at26.7-29.3 minutes, extracted into 40 mL n-butanol, and the solventremoved in vacuo to yield 34 mg of pure product. MS data=M+1 (FAB) 575,558, 541, 484.

EXAMPLE 4 Synthesis of 13-cyclopentylthio-5-proprionate-6-deoxytetracycline

100 mg of 13-cyclopentylthio-5-hydroxy-6-a-deoxy-tetracycline of Example2 and 5.0 g of propionic acid were dissolved in 35 mL of anhydroushydrofluoric acid and the resultant solution sealed in a polypropylenetube for 3 days at room temperature. The hydrofluoric acid was removedby a slow steady stream of nitrogen and the residue taken up in diethylether. The precipitate was dissolved in MeOH (4 mL) and injected into apreparative HPLC utilizing a C18 reverse-phase column and mobile phasesof phosphate buffer (pH 4.5) and MeOH over a linear gradient (30%-100%over 30 minutes) at 30 mL/minute. The compound was collected at26.7-29.3 minutes, extracted into 40 mL n-butanol, and the solventremoved in vacuo to yield 34 mg of pure product. MS data=M+1 (FAB) 601,492, 391.

The present invention is not to be restricted in form nor limited inscope except by the claims appended hereto.

I claim:
 1. A method for therapeutically treating a tetracyclineresistant cell with tetracyclines, which comprises the stepsofadministering to the cell a predetermined quantity of at least a firstcomposition selected from the chemical group consisting of a blockingagent which is capable of interacting with a product of at least onetetracycline resistance determinant capable of protecting ribosomes inthe cell from tetracycline's inhibitory activity; and concomitantlyadministering to the cell a predetermined quantity of at least a secondcomposition selected from the chemical group consisting of tetracycline,tetracycline analogues, and tetracycline derivatives which are not saidblocking agent.
 2. A method according to claim 1 wherein said blockingagent contains a sufficient part of tetracycline to interact with aproduct of at least one tetracycline resistance determinant capable ofprotecting ribosomes in the cell from tetracycline's inhibitoryactivity.
 3. A method according to claim 1 wherein said firstcomposition is present in a subinhibitory amount.
 4. A method accordingto claim 1 wherein said tetracycline resistance determinant belongs tothe Class A, B, K, L, M, O or Q tetracycline resistance determinant. 5.A method according to claim 1 wherein said blocking agent and saidsecond composition are employed in a molar ratio of from about 0.01 to100.
 6. A method according to claim 1 wherein said blocking agent isalso effective against a tetracycline efflux system.
 7. A methodaccording to claim 1 wherein said second composition is minocycline,doxycycline, methacycline, demeclocycline, oxytetracycline, orchlortetracycline.
 8. A method for converting tetracycline resistantbacteria into tetracycline sensitive bacteria, comprising contacting theresistant bacteria with a predetermined quantity of at least a firstcomposition selected from C5 esters of tetracycline, 13,5 derivative or6-deoxy-13-(substituted mercapto)tetracyclines, and concomitantlyadministering to the cell a predetermined quantity of at least a secondcomposition selected from a tetracycline, a tetracycline analogue or atetracycline derivative which is not a C5 ester of tetracycline nor a6-deoxy-13-(substituted mercapto)tetracycline.
 9. A method according toclaim 8 wherein said first composition is a 6-deoxy-13-(alkylsubstituted mercapto)tetracycline.
 10. A method according to claim 8wherein said first composition is a 6-deoxy-13-(aryl substitutedmercapto)tetracycline.
 11. A method according to claim 8 wherein saidfirst composition is a C5 ester.
 12. A method according to claim 8wherein said first composition is a 13,5 derivative.
 13. A methodaccording to claim 8 wherein said second composition is tetracycline.14. A method according to claim 8 wherein said second composition isminocycline, doxycycline, methacycline, demeclocycline, oxytetracycline,or chlortetracycline.
 15. A pharmaceutical preparation for convertingtetracycline resistant bacteria into tetracycline sensitive bacteriacomprising a blocking agent which is capable of interacting with aproduct of at least one tetracycline resistance determinant capable ofprotecting ribosomes in the cell from the inhibitory activity oftetracycline, a tetracycline type antibiotic, and a pharmaceuticalcarrier.
 16. A pharmaceutical preparation according to claim 15 whereinthe tetracycline-type antibiotic is a tetracycline, a tetracyclineanalogue or a tetracycline derivative.
 17. A pharmaceutical preparationaccording to claim 15 wherein said tetracycline resistance determinantbelongs to the Class A, B, K, L, M, O or Q tetracycline resistancedeterminants.
 18. A pharmaceutical preparation according to claim 15wherein the tetracycline-type antibiotic is selected from minocycline,doxycycline, methacycline, demeclocycline, oxytetracycline, orchlortetracycline.
 19. A pharmaceutical preparation for convertingtetracycline resistant bacteria into tetracycline sensitive bacteriacomprising a 6-deoxy-13(substituted mercapto)tetracycline, atetracycline-type antibiotic which is not a 6-deoxy-13-(substitutedmercapto)-tetracycline, and a pharmaceutically acceptable carrier.
 20. Apharmaceutical preparation for converting tetracycline resistantbacteria into tetracycline sensitive bacteria comprising a C5 ester oftetracycline, a tetracycline-type antibiotic which is not a C5 ester oftetracycline, and a pharmaceutically acceptable carrier.
 21. Apharmaceutical preparation for converting tetracycline resistantbacteria into tetracycline sensitive bacteria comprising a 13,5derivative of tetracycline, a tetracycline-type antibiotic which is nota 13,5 derivative of tetracycline, and a pharmaceutically acceptablecarrier.
 22. A pharmaceutical preparation according to claim 15 whereinthe tetracycline-type antibiotic is a tetracycline, a tetracyclineanalogue or a tetracycline derivative.
 23. A pharmaceutical preparationaccording to claim 15 wherein the tetracycline-type antibiotic isselected from minocycline, doxycycline, methacycline, demeclocycline,oxytetracycline, or chlortetracycline.
 24. A class of C5 esters oftetracycline compositions useful in combination with other classes oftetracyclines, tetracycline analogues and tetracycline derivatives, saidclass of compositions having the formula ##STR48## wherein R1 and R2 areselected from the group consisting of a methylene group, hydroxyl,hydrogen or a group consisting of organic entities comprising from 1-12carbon atoms, with or without other heteroatoms including sulfur,oxygen, halogen, nitrogen, and the like, and takes form as linear,branched, or cyclic alkyl, aryl, or alkylaryl structures; and A isselected from the group consisting of a hydrogen atom, a methylenegroup, and any linear, branched, or ring structure comprising from 1-6carbon atoms and optionally including heteroatoms such as oxygen andnitrogen atoms.
 25. The compositions of claim 24, wherein thecompositions are selected from the group of Formula II, wherein R₁ isCH₃, H and R₂ is COCH₂ CH₃, R₁ is ═CH and R₂ is COCH₂ CH₃, R₁ is --CH₂--S-cyclopentyl, H and R₂ is COCH₂ CH₃, or R₁ is --CH₂ --S-propyl and R₂is COCH₂ CH₃.
 26. A class of 6-deoxy-13-(substituted mercapto)tetracyline compositions useful in the therapeutic treatment of atetracycline resistant cell in combination with other classes oftetracyclines, tetracycline analogues and tetracycline derivatives, saidclass of compositions having the formula ##STR49## wherein A is hydrogenor hydroxyl, B comprises a morpholino group, andR is an organic entitycomprising 1-12 carbon atoms and optionally including heteratoms.
 27. Atetracycline composition according to claim 26 having the formula##STR50##
 28. The method of claim 1 wherein the first compositioncomprises 5-proprionate doxycycline and the second composition comprisesdoxycycline.